Flow cytometry is used to differentiate various types of cells and other similar small particles. Conventional flow cytometers commonly comprise an optically-transparent flow cell, usually made of quartz, having a central channel through which a stream of cells to be individually identified is made to flow. Movement of the cell stream through the flow cell channel is hydrodynamically entrained to the central longitudinal axis of the flow cell channel by a cell-free sheath liquid that concentrically surrounds the cell stream and flows along with the cell stream as it passes through the flow cell channel. As each cell passes through a cell-interrogation zone of the flow cell channel, it is irradiated with a focused beam of radiation (e.g., laser). Upon impinging upon each cell, the laser beam is scattered in a pattern characteristic of the morphology, density, refractive index and size of the cell. Further, the spectral characteristics of the laser beam may act to excite certain fluorochromes associated with selected cells, as may be the case when a cell's DNA has been previously stained with such fluorochromes, or when a fluorochrome molecule has been conjugated with a selected type of cell, either directly or via an intermediate. Photodetectors strategically positioned about the optical flow cell serve to convert the light-scattered by each cell and the fluorescence emitted by the excited fluorochromes to electrical signals which, when suitably processed, serve to identify the irradiated cell. In addition to the light scatter and fluorescence measurements made on each cell, some flow cytometers further characterize each cell by measuring certain physical and/or electrical properties of each cell as it passes through the flow cell. The cells may be sorted to selectively remove and collect certain cells of interest (e.g., abnormal cells) from the cells that have already passed through the optical flow cell and have been identified. Various sorting techniques have been developed, including methods requiring forming and deflecting droplets containing one or a small number of cells.
For example, a cell-sorting component may include a piezoelectric device that acts to vibrate the flow cell so as to effect the production a stream of droplets from the cell-entraining sheath liquid exiting from the flow cell. Ideally, each droplet contains but a single cell that has been characterized as to cell type by the light-scatter and fluorescence measurements just made on such cell. Each droplet in the droplet stream is then electrostatically charged as it passes between a pair of electrically charged plates, and each charged droplet is selectively deflected (or not deflected) towards a collection container as it passes between a pair of electrostatically charged deflection plates, such plates being charged to a droplet-deflecting polarity only at a time to deflect droplets (and cells) of interest. The instantaneous polarity of the deflection plates is determined by a cell-characterization processor that processes the cell-measurement signals from the optical flow cell.
Such sorting of microparticles such as cells is very important in biological research and medical applications. One of the first cell sorting apparatus was invented by Mack Fulwyler (e.g., U.S. Pat. No. 3,710,933). In his invention, tiny liquid droplets were sorted by electrostatic force. Most commercial cell sorters are still based on this technique, however other methods of cell sorting have been invented. Cells can be sorted by physical defection of cell stream, such as deflecting cell stream to desired channel with gas impulse (e.g., U.S. Pat. No. 4,175,662, U.S. Patent Application Publication No. 2011/0030808), by impulsive hydraulic force created by piezoelectric beam (U.S. Pat. No. 7,392,908), or by magnetostrictive gates (U.S. Pat. No. 7,160,730). Cells can also be sorted by manipulating single cells in micro-fabricated channels by optical force (U.S. Pat. No. 8,426,209, U.S. Pat. No. 7,745,221, U.S. Pat. No. 7,428,971, U.S. Patent Application Publication No. 2008/0138010), by acoustic force (U.S. Pat. No. 8,387,803, U.S. Patent Application Publication No. 2013/0192958, U.S. Patent Application Publication No. 2012/0160746), by magnetic force (U.S. Pat. No. 8,071,054, U.S. Pat. No. 7,807,454, U.S. Pat. No. 6,120,735, U.S. Pat. No. 5,968,820, U.S. Pat. No. 5,837,200), or by dielectrophoretic force (U.S. Pat. No. 8,454,813, U.S. Pat. No. 7,425,253, U.S. Pat. No. 5,489,506, U.S. Patent Application Publication No. 2012/0103817). All of these methods typically involve complex fluidic systems and sophisticated electrical control systems, which make cell sorting apparatus expensive to build, and difficult to use.
In addition, the vast majority of known cell sorting mechanisms are focused on the sorting mixed cells into two or more populations. Droplet sorting by electrostatic force as described in U.S. Pat. No. 3,710,933 is the preferred mechanisms to deliver sorted individual cell to a predetermined location in real time. Unfortunately, droplet sorting is typically limited to delivery of sorted individual cells to a relative large area (e.g., more than 5 mm in diameter); for areas smaller than 5 mm in diameter, the deliver accuracy becomes very low because droplets typically travel at speed more than 1 m/s and to aim the droplets precisely to an area less 5 mm in diameter (e.g., using electrostatic force) is very difficult, particularly the speed of droplet is not constant. For example, currently available droplet cell sorters, such as BD ARIA III can sort individual cells directly into 96-well cell culture plate, in which the area of each well is about 6.5 mm in diameter, with accuracy of 70%. However, sorting individual cell into 384-well cell culture plate which is about 3 mm in diameter is not practical.
In contrast, there are commercially available technologies for delivering small volumes of liquids to precise locations. For example, the FLEXDROP (Perkin Elmer) is a liquid dispenser capable of delivering small amounts of liquid to a precise location having an area of less than 1 mm in diameter. Unfortunately, such liquid dispensers cannot sort cells.
Thus, it would be beneficial to provide a microparticle sorter that can address the problems discussed above. In particular, it would be beneficial to provide methods and apparatus that are capable of automatically sorting microparticles (e.g., individual cells or small groups of cells in a liquid suspension) and delivering them in small volumes of liquid to small diameter wells. Described herein are apparatus and methods capable of easily, inexpensively and efficiently sorting microparticles.